Channelrhodopsins (ChR) are light-gated, non-specific cation channels that allow the selective depolarization of genetically targeted cells (Arrenberg, A. B. et al., Science 330, 971-974 (2010); Boyden, E. S. et al., Nat Neurosci 8, 1263-1268 (2005); Bruegmann, T. et al., Nat Methods 7, 897-900 (2010); Nagel, G. et al., Science 296, 2395-2398 (2002); Nagel, G. et al., Proc Natl Acad Sci USA 100, 13940-13945 (2003); and Adamantidis, A. R. et al., Nature 450, 420-424 (2007), the contents of which are hereby expressly incorporated by reference in their entireties for all purposes). As such, channelrhodopsins are used as genetically expressible proteins that are capable of depolarizing genetically selective neurons with high temporal and spatial precision. Currently available ChRs, however, are limited by action spectra that typically peak at 450-545 nm (Lin, J. Y. et al., Biophys J96, 1803-1814 (2009); Wen, L. et al., PLoS One September 23; 5(9) (2010); Govorunova, E. G. et al., MBio. June 21; 2(3) (2011); Kleinlogel, S. et al., Nat Neurosci 14, 513-518 (2011); and Yizhar, O. et al., Nature 477, 171-178 (2011), the contents of which are hereby expressly incorporated by reference in their entireties for all purposes).
In mammalian systems, these blue-green lights have limited penetration depths into tissue, as the light of these wavelengths are strongly absorbed by endogenous chromophores such as flavins, hemoglobin, and melanin. Blue-green light is also prone to a higher degree of scattering, as compared to light having longer wavelength, especially when penetrating through nervous tissues (Tromberg, B. J. et al., Neoplasia 2, 26-40 (2000)).
To circumvent this problem, one or more thin optical fibers can be inserted into neural tissue for deep ChR excitation (Aravanis, A. M. et al., J Neural Eng 4, 5143-156 (2007)). Although effective in eliciting ChR activation, such invasive procedures damage neural structures en route to the target, require precise stereotaxic positioning, may become damaged in freely behaving animals, and thus may be difficult to perform when ChR is expressed in deep nuclei, such as in the brainstem of mammals.
A number of channelrhodopsin variants are known in the art. For example, Lin et al. (Biophys J, 2009, 96(5): 1803-14) describe making chimeras of the transmembrane domains of ChR1 and ChR2, combined with site-directed mutagenesis. Zhang et al. (Nat Neurosci, 2008, 11(6): 631-3) describe VChR1, which is a red-shifted channelrhodopsin variant. VChR1 has lower light sensitivity and poor membrane trafficking and expression. Other known channelrhodopsin variants include ChR2 (Nagel, G., et al., Proc Natl Acad Sci USA, 2003, 100(24): 13940-5), ChR2/H134R (Nagel, G., et al., Curr Biol, 2005, 15(24): 2279-84), and ChD/ChEF/ChIEF (Lin, J. Y., et al., Biophys J, 2009, 96(5): 1803-14), which are activated by blue light (470 nm) but show no sensitivity to orange/red light. Additional variants have been disclosed by Lin (Lin, J. Y., Experimental Physiology, 2010, 96.1: 19-25). Knopfel et al. (The Journal of Neuroscience, 2010, 30(45): 14998-15004) have reviewed a number of second generation optogenetic tools, including ChR.